Unilateral inactivation of the basolateral amygdala attenuates context-induced renewal of Pavlovian-conditioned alcohol-seeking

Abstract

Environmental contexts associated with drug use promote craving in humans and drug-seeking in animals. We hypothesized that the basolateral amygdala (BLA) itself as well as serial connectivity between the BLA and nucleus accumbens core (NAC core) were required for context-induced renewal of Pavlovian-conditioned alcohol-seeking. Male Long-Evans rats were trained to discriminate between two conditioned stimuli (CS): a CS+ that was paired with ethanol (EtOH, 20%, v/v) delivery into a fluid port (0.2 mL/CS+, 3.2 mL per session) and a CS- that was not. Entries into the port during each CS were measured. Next, rats received extinction in a different context where both cues were presented without EtOH. At test, responding to the CS+ and CS- without EtOH was evaluated in the prior training context. Control subjects showed a selective increase in CS+ responding relative to extinction, indicative of renewal. This effect was blocked by pre-test, bilateral inactivation of the BLA using a solution of GABA receptor agonists (0.1 mm muscimol and 1.0 mm baclofen; M/B; 0.3 μL per side). Renewal was also attenuated following unilateral injections of M/B into the BLA, combined with either M/B, the dopamine D1 receptor antagonist SCH 23390 (0.6 μg per side) or saline infusion in the contralateral NAC core. Hence, unilateral BLA inactivation was sufficient to disrupt renewal, highlighting a critical role for functional activity in the BLA in enabling the reinstatement of alcohol-seeking driven by an alcohol context.

Keywords: addiction; ethanol; nucleus accumbens; pavlovian conditioning; reinstatement; relapse.

Fig 1. The acquisition and extinction of Pavlovian conditioned alcohol seeking

Data represent average (Mean ± SEM) responding in each session of Pavlovian discrimination training (PDT) in Context A and extinction (EXT) in context B. In each session subjects received 16 CS+ and 16 CS− presentations according to an independent VT-67 sec schedule. During PDT each CS+ was paired with 0.2 ml of 20% EtOH (3.2 ml total). A Normalized port entries made during the CS+ (filled circles) and the CS− (open circles) across session. Data were normalized to account for differences in baseline activity by subtracting port entries during the 10-seconds immediately before each CS from the corresponding CS. B Total port entries across session.

Fig 2. Bilateral BLA inactivation blocks context-induced renewal of Pavlovian conditioned alcohol seeking

For this and subsequent graphs, data represent average (Mean ± SEM) responding across the last 2 sessions of Pavlovian discrimination training (PDT) in Context A and extinction (EXT) in Context B, before the corresponding renewal test. At test in Context A subjects were pretreated with saline (filled bars) or M/B (gray bars) bilaterally into the BLA, using a within-subject design. They received CS+ and CS− presentations as during PDT, but no EtOH was delivered. A Normalized port entries during the CS+. For this and subsequent graphs filled circles represent a difference score obtained by subtracting normalized CS+ responding following M/B treatment from normalized CS+ responding after saline treatment. The horizontal bar represents the average of this difference score. B Normalized port entries during the CS−. C Total port entries. D Placement of injector tips within the BLA. For this and subsequent graphs distance from bregma is indicated to the right of each coronal section. ^ P = 0.003, BLA Saline EXT vs. BLA Saline TEST; * P = 0.002, BLA Saline TEST vs. BLA M/B TEST

Fig 3. Contralateral BLA – NAC core inactivation reduces context-induced renewal of Pavlovian conditioned alcohol seeking

Before the renewal test in Context A subjects received unilateral infusions of either saline (filled bars) or M/B (gray bars) into the contralateral BLA and NAC core, using a within-subjects design. A Normalized port entries during the CS+. B Normalized port entries made during the CS−. C Total port entries. D Placement of injector tips within the BLA. E Placement of injector tips within the NAC core. ^ P < 0.001 BLA-NAC core Saline EXT vs. BLA-NAC core Saline TEST; * P = 0.001 BLA-NAC core Saline TEST vs. BLA-NAC core M/B

Fig 4. Context-induced renewal of Pavlovian conditioned alcohol seeking is reduced by unilateral BLA inactivation, and by unilateral BLA inactivation with unilateral SCH 23390 in the contralateral NAC core

Treatment conditions at test are indicated in the legend. A Normalized port entries during the CS+. B Normalized port entries made during the CS−. C Total port entries. D Placement of injector tips within the BLA. E Placement of injector tips within the NAC core. ^ P < 0.001, BLA-NAC core Saline EXT vs. BLA-NAC core Saline TEST; * P = 0.03, significant difference between BLA saline–NAC core saline vs. BLA M/B–NAC core SCH 23390; * P = 0.04, significant difference between BLA saline–NAC core saline vs. BLA M/B–NAC core saline.

Fig 5. No effect of unilateral BLA inactivation or contralateral BLA M/B - NAC core SCH23390 on Pavlovian conditioned alcohol seeking

At test rats received PDT sessions in Context A where CS+ presentations were paired with EtOH. Samples sizes for each treatment condition as the same as in Figure 4. Data represent average (Mean ± SEM) normalized port entries during the CS+ and CS−. Treatment conditions at test are indicated in the legend.